CN106432385A - Preparation method for high-purity breviscapine extract as well as preparations and application thereof - Google Patents
Preparation method for high-purity breviscapine extract as well as preparations and application thereof Download PDFInfo
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- CN106432385A CN106432385A CN201510589982.7A CN201510589982A CN106432385A CN 106432385 A CN106432385 A CN 106432385A CN 201510589982 A CN201510589982 A CN 201510589982A CN 106432385 A CN106432385 A CN 106432385A
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- methanol
- breviscapine
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Abstract
The invention provides a preparation method for high-purity breviscapine extract as well as preparations and application thereof. The method disclosed by the invention comprises the following steps: taking erigeron breviscapus as a raw material, performing dynamic ultrasonic countercurrent extraction or heating reflux extraction, filtering, centrifuging, adjusting the pH value to 2.0-7.0, performing macroporous resin column chromatography, preparing by using dynamic axial compression industrial chromatographic separation, or preparing by adopting high-speed counter-current chromatography, freezing or performing spray drying, thereby obtaining the high-purity breviscapine extract. According to the method disclosed by the invention, extraction can be completed in a short time at a low temperature, the separation cycle is short, the efficiency is high, the occupied volume for extraction is reduced, the production cost is lowered, the energy consumption and environmental pollution can be reduced, and the purity of the obtained breviscapine can be 95% or higher. Added with various added pharmaceutical adjuvants, the high-purity breviscapine extract can be prepared into ordinary tablets, thin membrane coated tablets, capsules, dispersible tablets, dropping pills, granules, sustained-release preparations and other solid oral preparations, as well as spray, aerosol, lyophilized powder for injection and injection, and the preparations can be used for treating cardiovascular and cerebrovascular diseases and are obvious in curative effects, safe and reliable.
Description
Technical field
The invention belongs to plant and Chinese medicine extract field, particularly to a kind of high-purity breviscapine extract and preparation
Preparation method and use.
Background technology
Cardiovascular and cerebrovascular disease is the number one killer of serious harm human health and life in the world today, its sickness rate and dead
Rate of dying exceedes malignant tumor and leaps to No. 1 in the world.According to World Health Organization (WHO) (WHO) report, annual 17000000 people in the whole world
Die from cardiovascular and cerebrovascular disease, account for the 30.3% of dead population, estimate that the year two thousand thirty whole world cardiovascular and cerebrovascular disease death toll will reach
To 23,300,000【Go AS,Mozaffarian D,Roger VL,Benjamin EJ,Berry JD,Borden WB,et
al.Heart disease and stroke statistics--2013update:a report from the American
Heart Association.Circulation.2013；127(1):e6-e245】.Defend planning commission's statistics, China city according to country
Human mortality highest is cardiovascular and cerebrovascular disease.National cardiovascular and cerebrovascular vessel vascular disease 2.3 hundred million people, dies from heart and brain blood every year
The total number of persons of pipe disease is 2,600,000 people, has 300 people to be seized life by cardiovascular and cerebrovascular disease per hour.And the morbidity of this disease
In ascendant trend year by year, the age is in rejuvenation to rate.At present, cardiovascular and cerebrovascular disease medication has become the first big of world's medical market
Quasi drugss.
Herba Erigerontiss are commonly called as Herba Erigerontiss, are Compositae Herba Erigerontis aceris platymiscium Erigeron breviscapus (Vant.) Hand.-Mazz. (Erigeron breviscapus
(Vant.) Hand.-Mazz) herb is dried, be distributed mainly on Yunnan Province of China, Sichuan, Guizhou etc. save, especially produced with Yunnan
Herba Erigerontiss are well-known, and yield accounts for the 90% of the whole nation, have activating blood circulation to dissipate blood stasis, relaxing muscles and tendons, pain relieving, improve microcirculatory special efficacy
【Lei Ting, Wang Jianchao, Liu Guangming. Advance on Pharmacological Activities in cardiovascular and cerebrovascular disease for the breviscapine, Medical review, 2009,
15 (18) : 2844-2846】.Breviscapine is the flavonoids effective constituent extracting from short booth Herba Erigerontis aceris, predominantly Herba Erigerontiss
B prime (Breviscarpine) and breviscapine (4 ', 5 dihydroxyflavone -7-O- Fructus Vitis viniferaes
Saccharic acid glycosides) (structure is shown in formula 1).Wherein, lamp-dish flower acetic is its main active, content>95%.Pharmaceutical research shows,
Breviscapine has expansion of cerebral vascular, reduces cerebral vascular resistance, increases cerebral blood flow, improves microcirculation；Antiplatelet aggregation,
Anticoagulation, inhibition thrombosis, improve hemorheological property【Lu J, Cheng C, Zhao X, et al. PEG-
scutellarin prodrugs: synthesis, water solubility and protective effect on
cerebral ischemia/reperfusion injury.Eur JMed Chem.2010; 45 (5): 1731-
1738】；Suppression neuronal apoptosis and the toxicity of excitatory amino acid (EAA), suppress inflammatory reaction, mitigate blood brain barrier and damage
Wound is hence it is evident that increase cerebral ischemia and reperfusion tissues following MCAO in rats Na+-K+～ATP enzyme and Ca2+～atpase activity, improves cerebral tissue energy
Metabolism, mitigates cerebral edema【Zhang HF, Hu XM, Wang LX, et al. Protective effects of
scutellarin against cerebral ischemia in rats: evidence for inhibition of the
apoptosis-inducing factor pathway.Planta Med.2009; 75 (2): 121-126】；Notable increasing
Plus coronary flow, reduce myocardial infarct size, anti-lipid peroxidation, mitigate radical damage, suppress Protein kinase C, subtract
Gently intracellular Ca2+Overload, improves the effect such as body hypoxia-bearing.At present, clinic is mainly used in the treatment of cardiovascular and cerebrovascular disease, curative effect
Definitely, to coronary heart diseases and angina pectoris, acute myocardial infarction, cerebral thrombosiss, cerebral blood supply insufficiency, apoplexy sequela, ischemic cerebrovascular
Acute stage, the symptom such as paralysis caused by convalescent period and cerebral infarction, total effective rate reaches more than 90%.Simultaneously can be used for occlusive
Angiopathy, vasculitiss, chronic renal failure, diabetes and its complication, nephrotic syndrome, chronic pulmonary heart disease etc.
Treatment.
Lamp-dish flower acetic（Scutellarin）Herba Erigerontiss A prime（Apigenin-7-O-glucuronide）
Formula 1 lamp-dish flower acetic and Herba Erigerontiss A prime structure
Breviscapine and Breviscapine are by 2015 editions《Chinese Pharmacopoeia》Include, there is nearly various schools of thinkers pharmacy corporation in the whole nation
Produce Breviscapine, yield is in cumulative year after year trend.As a kind of important plant amedica intermediate, its extract is also used for
Outlet, the raw material of breviscapine and preparation annual value of production have broken through 10,000,000,000 yuan.At present, the extraction and separation process road of existing breviscapine
Line is excessively coarse, leads to that the purity of product Main Ingredients and Appearance is low, and impurity content is higher, some Breviscapini injection Clinical practice
Afterwards, accidental uncomfortable in chest, the weak, untoward reaction such as erythra, itch all over, cardiopalmus.Therefore, for existing extraction and separation process not
Foot, seeks a kind of steady quality, the preparation method of high-purity breviscapine of polishing purification rational technology and its related preparations
Research and development, have important clinical treatment meaning and wide market prospect.
Content of the invention
It is an object of the invention to provide a kind of extracted or heating and refluxing extraction using dynamic ultrasound adverse current, inhale in conjunction with macropore
Attached resin column chromatography, dynamic axial compression process-scale chromatography or high speed adverse current chromatogram separate prepares high-purity breviscapine extract
Method, this technical process is simple, quality controllable.The high-purity breviscapine prepared in this way, can be medicinal by adding
Medically acceptable various dosage forms made by adjuvant, for clinical treatment coronary heart diseases and angina pectoris, acute myocardial infarction, cerebral thrombosiss,
Paralysis caused by cerebral blood supply insufficiency, apoplexy sequela, the acute stage of ischemic cerebrovascular, convalescent period and cerebral infarction.Simultaneously
Can be used for obliterative vascular disease, vasculitiss, chronic renal failure, diabetes and its complication, nephrotic syndrome, chronic pulmonary
The symptoms such as source property heart disease.
The inventive method is realized by following steps：Fourth Herba Erigerontiss are crushed to 10～20 mesh, plus 6～20 times of volume of water
Or the alcoholic solution that concentration of volume percent is 10%～80%, or acetone soln, or aqueous isopropanol, dynamic ultrasound adverse current extract or
Heating and refluxing extraction 2～3 times, 0.5～2h every time, filters, united extraction liquid, recovered under reduced pressure Extraction solvent, is suitably concentrated into dense
Spend for 0.4～2.0 g crude drug/ml, centrifugation or after filtering, plus dilute hydrochloric acid adjust pH to 2.0～7.0 upper prop solution, upper prop is molten
Liquid passes through macroporous adsorption resin chromatography post with the flow velocity of 2BV/h～4BV/h, and upper prop solution is according to the ratio of solid content and dried resin
For 1：5～10, first with the distilled water washing resin post bed of 4～6BV, elution flow rate is 2BV/h～4BV/h, discards water elution；
The alcohol eluent being 10%～80% with concentration of volume percent again, elution flow rate is 2BV/h～4BV/h, merges eluent
Recovered under reduced pressure alcohol, being evaporated to relative density is 1.05～1.30, adds the column chromatography silica gel filler of 200～300 mesh, plus first
Post-drying mixed thoroughly by alcohol, crosses the reverse industrial preparation chromatographic column of dynamic axial compression, and methylene chloride-methanol, chloroform-methanol or methanol-
Water (6：1～2：1) it is eluent gradient eluting, collect the principal piece eluent containing breviscapine respectively, merge, concentrating under reduced pressure, plus
Enter 10 times amount methylene chloride-methanols（8：1～5：1）Recrystallization, filters, decompression and solvent recovery, lyophilization, is spray-dried or subtracts
Press dry dry, obtain final product high-purity breviscapine（HPLC detects purity >=95%）.
Or take the macroporous resin crude extract of Herba Erigerontiss, add n-hexane-ethyl acetate-methanol/ethanol-water system or just
In the lower phase of hexane-ethylacetate-n-butanol-water system, concussion makes to be completely dissolved, and carries out high speed adverse current chromatogram (HSCCC) point
From preparation.High speed adverse current chromatogram separation condition：Detection wavelength is 335 nm, and main frame temperature is 25 DEG C, n-hexane-ethyl acetate-
Methanol-water is solvent system or n-hexane-ethyl acetate-n-butanol-water system（2：8：4：4～6 or 3：7：3～4：6～8）,
N-hexane-ethyl acetate（Fixing phase）For upper phase, methanol/ethanol-water or n-butanol-water（Mobile phase）For lower phase.By fixing phase
Fill chromatography column with 10～30 ml/min flow velocitys, adjustment high-speed counter-current chromatograph engine speed is 600～900 rpm/
Min, enters mobile phase with the flow pump of 2.0～6.0 ml/min simultaneously, treat biphase reach dynamic equilibrium, open injection valve, sample
Liquid sample introduction, gathered data simultaneously records ultraviolet chromatogram, reclaims purpose flow point, decompression and solvent recovery, freezing under 335 nm wavelength
Or be spray-dried, obtain high-purity breviscapine extract（HPLC detects purity >=95%）.
Macroporous adsorbent resin of the present invention is polarity, low pole and non-polar macroporous resin in polystyrene type,
Including ADS-17, ADS-750, LSA-21, LX-26, XDA-6, D101, D113, D151, D152, D3520, H103, HPD100A,
HPD-300、HPD400、HPD500、HPD-650、HPD-700、HPD-750、HPD-800、HPD-820、DM301、X-5、S-8、
SP825, CAD45,860021, NKA- II or NKA-9 one or more, preferably macroporous adsorbent resin D152, HPD-400 or
HPD-650 type.During centrifugation, centrifugal speed is in the range of 3000～10000 revs/min.Dynamic axial compression (DAC) industry
Preparative hplc filler is the column chromatography silica gel of 100～300 mesh, and elution flow phase is methylene chloride-methanol, chloroform-methanol or first
Alcohol-water system (6：1～2：1) gradient elution.Eluting solvent used by high speed adverse current chromatogram (HSCCC) be n-hexane-ethyl acetate-
Methanol/ethanol-water system or n-hexane-ethyl acetate-n-butanol-water system.
It is an advantage of the invention that：
1. the present invention utilizes dynamic ultrasound adverse current extraction process first, in conjunction with macroporous adsorbent resin column chromatography and dynamic axial compression
Preparing chromatograph in industry or high speed adverse current chromatogram isolation technics, prepare high purity more than 95% breviscapine extract, and can lead to
Cross and add various pharmaceutic adjuvants and make multiple dosage forms, for developing treatment cardiovascular and cerebrovascular disease, obliterative vascular disease, vascular
The Chinese medicine five of the disease such as inflammation, chronic renal failure, diabetes and its complication, nephrotic syndrome, chronic pulmonary heart disease
Kind new medicine provides material base；
2. the present invention is reasonable in design, process is simple, is followed the example of using dynamic ultrasound alcohol extraction, and macroporous adsorbent resin column chromatography combines dynamic
It is compressed axially process-scale chromatography or high speed adverse current chromatogram isolation technics, obtain highly purified breviscapine extract, non-environmental-pollution,
Simple and efficient to handle, cost is relatively low, is especially suitable for industrialized production；
3. the purification with macroreticular resin method selected by the present invention, has adsorbance greatly, desorption efficiency is high, can be anti-after resin regeneration
The advantages of use again；Dynamic axial compression (DAC) preparing chromatograph in industry is a kind of efficient isolation technics, with traditional preparative hplc
Compare, there is the features such as load sample amount is big, and the filling filler time is short, chromatogram column efficiency is high, separating rate is fast, can reach close point
The post effect of analysis post；High speed adverse current chromatogram isolation technics (HSCCC) have that carrier-free rapidly and efficiently separates, the response rate is high, fractional dose
Greatly, simple operation and other advantages, are widely used in the separation preparation of active skull cap components in recent years, well should therefore have
Use prospect.
Obtained high-purity breviscapine extract, can be made into conventional tablet, thin film by adding various pharmaceutic adjuvants
The solid orally ingestibles such as coated tablet, capsule, dispersible tablet, drop pill, granule, sustained-release preparation, and spray, aerosol
The dosage forms such as agent, freeze-dried powder, injection.
Brief description
Fig. 1 is the preparation technology flow chart of high-purity breviscapine extract of the present invention and preparation.
Fig. 2 is the HPLC chromatogram of high-purity breviscapine extract.
Fig. 3 is the triangle dispersible tablet prepared for raw material using high-purity breviscapine.
Specific embodiment
The invention will be further described with reference to embodiments, but the invention is not limited in these embodiments.
Embodiment 1 dynamic ultrasound adverse current is extracted industrially prepared with reference to macroporous adsorbent resin column chromatography and dynamic axial compression
Chromatographic separation technology prepares high-purity breviscapine extract
Herba Erigerontiss medical material 10 kg is crushed to 20～30 mesh, plus the ethanol that 8 times amount concentration of volume percent are 70%, in frequency
30 kHz, ultrasonic power 20kW Continuous Countercurrent Extraction 3,40 min every time, filter, united extraction liquid, recovered under reduced pressure Extraction solvent,
Suitably being concentrated into concentration is 0.6 g crude drug/ml, plus 1 mol/L hydrochloric acid adjusts pH value to 4, passes through HPD650 with the flow velocity of 2BV/h big
Macroporous adsorbent resin chromatographic column, sample solution is 1 in the ratio of solid quality and dried resin：6, blade diameter length ratio is 1：8, first with 4 BV's
Distilled water washing resin post bed, elution flow rate is 2 BV/h, discards water elution；The second being 50% with concentration of volume percent again
Alcohol eluting, elution flow rate is 2 BV/h, eluent decompression recycling ethanol, and being concentrated into relative density is 1.15 (50 DEG C record), plus
Enter the column chromatography silica gel filler of 200～300 mesh, plus methanol mixes post-drying thoroughly, crosses the reverse preparing chromatograph in industry of dynamic axial compression
Post, methylene chloride-methanol (5：1～3：1) it is eluent gradient eluting, collect the principal piece eluent containing breviscapine respectively, close
And, concentrating under reduced pressure, add 10 times amount methylene chloride-methanols（8：1）Recrystallization, filters, decompression and solvent recovery, lyophilization or spray
Mist is dried, and records lamp-dish flower acetic content >=95%（HPLC method, percentage by weight）.
Embodiment 2 heating and refluxing extraction separate with high speed adverse current chromatogram with reference to macroporous adsorbent resin column chromatography prepare high-purity
Degree breviscapine extract
In the 2kg Herba Erigerontiss medical material being crushed to 20～30 mesh, plus the ethanol that 15 times amount concentration of volume percent are 80%, plus
Circumfluence distillation 3 times, 1.5 h every time, filters, united extraction liquid, being concentrated into concentration is 1.0 g crude drugs/ml, Jia 1 after filtration
Mol/L hydrochloric acid adjusts pH value to 4～5, passes through HPD400 macroporous adsorption resin chromatography post with the flow velocity of 3BV/h, sample solution presses solid
The ratio of amount of substance and dried resin is 1：8, blade diameter length ratio is 1：10, first with the water washing resin of 6 BV, elution flow rate is 4 BV/h,
Discard water elution；The methanol-eluted fractions being 60% with concentration of volume percent again, elution flow rate is 3 BV/h, and eluent reduces pressure back
Receive methanol, being concentrated into relative density is 1.25 (50 DEG C record), drying under reduced pressure adds n-hexane-ethyl acetate-methanol-water system
In the lower phase of system, concussion makes to be completely dissolved, and carries out high speed adverse current chromatogram (HSCCC) and separates preparation.High speed adverse current chromatogram lightning strip
Part：Detection wavelength is 285 nm, and main frame temperature is 25 DEG C, and n-hexane-ethyl acetate-methanol-water is solvent system（2：8：4：4
～6 or 3：7：3～4：6～8）, n-hexane-ethyl acetate（Fixing phase）For upper phase, methanol/ethanol-water or n-butanol-water（Stream
Dynamic phase）For lower phase.Fixing phase is filled chromatography column with 10 ml/min flow velocitys, adjusts high-speed counter-current chromatograph engine speed
For 900 rpm/min, mobile phase is entered with the flow pump of 2.0 ml/min simultaneously, treat biphase reach dynamic equilibrium, open injection valve,
Sample liquid sample introduction, gathered data simultaneously records ultraviolet chromatogram, recovery purpose flow point under 335 nm wavelength, decompression and solvent recovery,
Freezing or spray drying, record lamp-dish flower acetic content 95%（HPLC method, percentage by weight）.
Embodiment 3 heating and refluxing extraction is divided with reference to macroporous adsorbent resin column chromatography and dynamic axial compression preparing chromatograph in industry
Prepare high-purity breviscapine extract from technology
Herba Erigerontiss 5 kg appropriateness is pulverized, plus 60% methanol of 10 times amount, in frequency 30 kHz, ultrasonic power 20kW continuous ultrasound
Adverse current is extracted 3 times, 0.5 h every time, filters, united extraction liquid, recovered under reduced pressure methanol, and the regulation concentration that adds water to 1.5 g crude drugs/
Ml, plus 1 mol/L hydrochloric acid adjusts pH value to 6, passes through SP825 macroporous adsorption resin chromatography post 2 times, sample solution with the flow velocity of 2BV/h
It is 1 in the ratio of solid quality and dried resin：9, blade diameter length ratio is 1：8, first with the water washing resin of 4 BV, elution flow rate is 2
BV/h, discards water elution；The ethanol elution being 70% with concentration of volume percent again, elution flow rate is 3 BV/h, and eluent subtracts
Push back receipts ethanol, being concentrated into relative density is 1.15 (50 DEG C record), adds the column chromatography silica gel filler of 200～300 mesh, plus
Methanol mixes post-drying thoroughly, crosses the reverse industrial preparation chromatographic column of dynamic axial compression, chloroform-methanol (6：1～4：1) it is mobile phase ladder
Degree eluting, collects the principal piece eluent containing breviscapine respectively, merges, concentrating under reduced pressure, add 10 times amount methylene chloride-methanols
（5：1）Recrystallization, filters, decompression and solvent recovery, lyophilization or spray drying, records lamp-dish flower acetic content 92%（HPLC
Method, percentage by weight）.
Embodiment 4 dynamic ultrasound adverse current is extracted and is separated preparation with high speed adverse current chromatogram with reference to macroporous adsorbent resin column chromatography
High-purity breviscapine extract
Herba Erigerontiss 5kg appropriateness is pulverized, plus 50% ethanol of 12 times amount, and in frequency 30 kHz, ultrasonic power 20kW continuous ultrasound is inverse
Stream extracts 3 times, 1.5 h every time, filters, united extraction liquid, decompression recycling ethanol, adds water and adjusts concentration to 1.2 g crude drugs/ml,
Plus 1 mol/L hydrochloric acid adjust pH value to 3～5, D152 macroporous adsorption resin chromatography post 2 times is passed through with the flow velocity of 3BV/h, sample solution is pressed
The ratio of solid quality and dried resin is 1：10, blade diameter length ratio is 1：8, first with the water washing resin of 4 BV, elution flow rate is 2
BV/h, discards water elution；The ethanol elution being 50% with concentration of volume percent again, elution flow rate is 3 BV/h, and eluent subtracts
Push back receipts ethanol, being concentrated into relative density is 1.15 (50 DEG C record), drying under reduced pressure, addition n-hexane-ethyl acetate-methanol-
In the lower phase of water system, concussion makes to be completely dissolved, and carries out high speed adverse current chromatogram (HSCCC) and separates preparation.High speed adverse current chromatogram divides
From condition：Detection wavelength is 285 nm, and main frame temperature is 25 DEG C, and n-hexane-ethyl acetate-methanol-water is solvent system（3：7：
3～4：6～8）, n-hexane-ethyl acetate（Fixing phase）For upper phase, methanol/ethanol-water or n-butanol-water（Mobile phase）For under
Phase.Fixing phase is filled chromatography column with 20 ml/min flow velocitys, adjustment high-speed counter-current chromatograph engine speed is 600 rpm/
Min, enters mobile phase with the flow pump of 3.0 ml/min simultaneously, treat biphase reach dynamic equilibrium, open injection valve, sample liquid is entered
Sample, gathered data simultaneously records ultraviolet chromatogram, reclaims purpose flow point, decompression and solvent recovery, freezing or spray under 335 nm wavelength
Mist is dried, and records lamp-dish flower acetic content 90%（HPLC method, percentage by weight）.
Embodiment 5
Herba Erigerontiss 5 kg appropriateness is pulverized, plus 30% ethanol of 13 times amount, heating and refluxing extraction 3 times, and 2.0 h every time filters, and closes
And extracting solution, decompression recycling ethanol, the regulation concentration that adds water is to 0.8 g crude drug/ml, plus 1 mol/L hydrochloric acid adjusts pH value to 6.5, with
The flow velocity of 2BV/h passes through HPD700 macroporous adsorption resin chromatography post 2 times, and sample solution in the ratio of solid quality and dried resin is
1：6, blade diameter length ratio is 1：8, first with the water washing resin of 4 BV, elution flow rate is 2 BV/h, discards water elution；Use volume hundred again
Divide the ethanol elution that specific concentration is 70%, elution flow rate is 3 BV/h, eluent decompression recycling ethanol, and being concentrated into relative density is
1.20 (50 DEG C record), add the column chromatography silica gel filler of 200～300 mesh, plus methanol mixes post-drying thoroughly, crosses dynamic axial pressure
Contract reverse industrial preparation chromatographic column, methylene chloride-methanol (5：1～2：1) it is eluent gradient eluting, collect respectively and contain Herba Erigerontiss
The principal piece eluent of element, merges, concentrating under reduced pressure, adds 10 times amount methylene chloride-methanols（6：1）Recrystallization, filters, recovered under reduced pressure
Solvent, lyophilization or spray drying, record lamp-dish flower acetic content 88%（HPLC method, percentage by weight）.
Embodiment 6
Herba Erigerontiss 5 kg appropriateness is pulverized, plus 20% ethanol of 10 times amount, heating and refluxing extraction 2 times, and each 2h filters, and merging carries
Take liquid, decompression recycling ethanol, the regulation concentration that adds water is to 0.5 g crude drug/ml, plus 1 mol/L hydrochloric acid adjusts pH value to 7.0, with 3BV/h
Flow velocity pass through 860021 macroporous adsorption resin chromatography post 2 times, the ratio that sample solution presses solid quality and dried resin is 1：6,
Blade diameter length ratio is 1：8, first with the water washing resin of 4 BV, elution flow rate is 2 BV/h, discards water elution；Use percent by volume again
Concentration is 60% ethanol elution, and elution flow rate is 3 BV/h, eluent decompression recycling ethanol, and being concentrated into relative density is 1.20
(50 DEG C record), adds the column chromatography silica gel filler of 200～300 mesh, plus methanol mixes post-drying thoroughly, crosses dynamic axial compression reverse
Industrial preparation chromatographic column, methylene chloride-methanol (5：1) it is mobile phase eluting, collect the principal piece eluent containing breviscapine respectively,
Merge, concentrating under reduced pressure, add 15 times amount methylene chloride-methanols（7：1）Recrystallization, filter, decompression and solvent recovery, lyophilization or
It is spray-dried, record lamp-dish flower acetic content 76%（HPLC method, percentage by weight）.
The preparation of high-purity breviscapine extract formulation
The preparation of embodiment 7 breviscapine drop pills
High-purity breviscapine extract 25 g, adds melting mixing substrate PEG6000 of 75 g：PEG4000 (1:1), stir
It is transferred in reservoir after uniformly, airtight and be incubated at 75 DEG C, with dosing pump pill dripping machine dripping from top to bottom, coolant is diformazan
Base silicone oil, water dropper bore（Inside/outside footpath）：2.1/2.5 mm, drips speed：30 balls/minute, drip away from：8 cm, every ball 25mg, collect and drip
Ball, sucks surface coolant, is dried, obtains final product.
The preparation of embodiment 8 erigeron breviscapus dispersion tablet
Dispersion tablet recipe：
Crude drug 80 g (20%)
Lactose 50 g（12.5%）
Amylum pregelatinisatum 102.5 g (25.63%)
Microcrystalline Cellulose（MCC） 100 g (25%)
Carboxymethyl starch sodium（CMS-Na） 27.5 g (6.87%)
Crospovidone (PVPP) 37.5 g (9.38%)
Micropowder silica gel 2.5 g (0.63%)
95% ethanol solution is appropriate
Make 1000 (0.4 g/ pieces)
Take high-purity breviscapine extract 80g, with amylum pregelatinisatum 102.5 g, Microcrystalline Cellulose 100g, Lactose 50 g, carboxylic
Methyl starch sodium 27.5 g, Crospovidone 37.5 g mix, plus 95% appropriate amount of ethanol makes soft material, and 24 mesh sieves are pelletized, 60 DEG C
It is dried, after adding micropowder silica gel 2.5 g to mix, 22 mesh sieve granulate, tabletted, piece type is triangle Special-shaped sheet, and piece weighs 400
Mg, the every mg containing lamp-dish flower acetic 80.
The preparation of embodiment 9 breviscapine injection freeze-dried powder
High-purity breviscapine 15 g, adds 20g Mannitol, injects water to 200ml dissolving, with the NaOH solution of 1mol/L
Adjust pH to 7.5, add 0.5% activated carbon, keep pH to 7.5, heating micro-boiling 15 minutes, be stirred continuously in the process, be cooled to
50 DEG C about filtration, be cooled to after room temperature through 0.45 μm of filtering with microporous membrane, be sub-packed in the cillin bottle of 5ml by every bottle of 2ml,
115 DEG C of flowing steam sterilizations 30 minutes, lyophilization, take out rear pressing cover packaging and obtain final product.
The preparation of embodiment 10 breviscapine capsule
Take high-purity breviscapine 40g, mix with Lactose 20g, starch 60g, plus soft material made by 95% ethanol, pelletized with 14 mesh sieves
Afterwards, put 60 DEG C～70 DEG C dryings after 12 mesh sieve granulate, be inserted in No. 1 Capsuleses and obtain final product, every capsule medicated 80 mg.
The preparation of embodiment 11 breviscapine conventional tablet
Take high-purity breviscapine 25 g, mix with starch 45 g, Microcrystalline Cellulose 15g, Lactose 12 g, carboxymethyl starch sodium 8g
Even, plus 10% starch slurry makes soft material in right amount, adds micropowder silica gel 0.5g, after mixing, 30 mesh sieves are pelletized, 24 mesh sieve granulate, compacting
In flakes, theoretical piece weight 400mg, every pastille 100 mg.
The pharmacology activity research of high-purity breviscapine extract
The protective effect to rat cerebral ischemia for the embodiment 12 high-purity breviscapine extract
1. experiment material
1.1 Experimental agents
Breviscapine extract (self-control, purity ＞ 95%), Nimodipine Tablets (Shanxi Yabao Pharmaceutical Group Corp., lot number
090806, every 20 mg).
1.2 laboratory animal
Healthy male SD rat, body weight：180-220 g；（Shanghai Slac Experimental Animal Co., Ltd. provides）
1.3 experiment reagent
Chloral hydrate (upper seamount Pu chemical company)
NaCl injection (Pharmaceutical Factory No.6, Harbin Pharmaceutical Group)
10% chloral hydrate is prepared：Weigh 10 g chloral hydrate, be dissolved in 100 ml normal saline.
2,3, 5-Triphenyltertrazoliumchloride (TTC), Sigma Co., USA
Superoxide dismutase (SOD) test kit, malonaldehyde (MDA) test kit, nitric oxide (NO) test kit（Life is built up in Nanjing
Thing Graduate School of Engineering）
2. laboratory animal packet：
Healthy SD rat 60, body weight：180-220g.Buy rear adaptability to raise 7 days, be randomly divided into 6 groups, every group 10, that is,
Sham operated rats, model group, nimodipine matched group（20 mg/kg）, breviscapine high dose group（40 mg/kg）, breviscapine
Middle dose group（20 mg/kg）, breviscapine low dose group（10 mg/kg）, daily gastric infusion 2 times, continuous 7 days.Sham-operation
Group and model group give equal-volume 0.5% carboxymethylcellulose sodium solution.
3. middle cerebral artery thromboembolism global cerebral ischemia-reperfusion (ischemia-reperfusion, IR) Establishing an injured model：
Method preparation IR model [1,2] with reference to Longa etc..By SD rat 10% chloral hydrate（350 mg/kg）Lumbar injection
Anesthesia, dorsal positions are fixed in laboratory table.Take neck median line otch, separate left common carotid（CCA）, external carotid artery（ECA）
And internal carotid artery（ICA）.The nylon wire of a diameter of 0.23 mm is inserted ICA through CCA, inlet wire depth is 16～19 mm.Block
Left side blood flow of middle cerebral artery 2 h, extracts nylon wire and gives Reperfu- sion 4 h.Sham operated rats only separate common carotid artery, external carotid artery
And internal carotid artery, not inaccessible middle cerebral artery.In art, strict control room temperature is at 25 DEG C about.
4. function of nervous system's sign is evaluated
Experiment modeling regain consciousness after and administration latter 1 day, 3 days, 7 days equal time points neurological deficits score is carried out to every rat,
All detections are completed by unwitting same person-time to rat packet, according to MACO rat behavior standards of grading [1-3], carry out
5 points of Neuroscore processed, fraction is higher, points out laboratory animal nerve injury more serious.Standards of grading are as follows：
Table 1 Neuroscore standard
| Laboratory animal active situation | Scoring | Neurological functional deficit |
| Limb activity is normal | 0 | Impassivity damages sign |
| It is unable to forelimb on the right side of full extension | 1 | Slight focal lesion |
| There is right side rotation during walking | 2 | Moderate focal lesion |
| Right side is had to topple over during autonomic movement | 3 | Severe focal lesion |
| No autonomic activitieses with disturbance of consciousness | 4 | Pole severe injury |
5. TTC dyeing measures rat cerebral infarction area percentage；
5.1 TTC dyeing
Rat sacrificed by decapitation after scoring is learned by function of nervous system, takes rapidly brain, and normal saline rinses and freezes 30 in rearmounted -20 DEG C of refrigerators
Min, excision olfactory bulb, cerebellum and low brain stem, interval 3 mm continuously do cerebral tissue coronal section.Cerebral tissue coronal section is put
The 2 of 2%, 3,5 tetrazolium chlorides（Triphenyl tetrazolium chloride, TTC）37 DEG C of lucifuges in normal saline solution
Temperature bath 30min, period is stirred every 10 min and once makes brain piece even dyeing.TTC can be reduced by mitochondrion catalase, raw
Become red, photosensitive fat-soluble Formazen, normal cerebral tissue is dyed redness, ischemic infarction area presents white because of this reaction of nothing
Color.
The calculating of 5.2 rat cerebral infarction area percentages
Brain tissue slice after TTC dyeing 30min, fix and divided with Image/J image after image scanning by 4% paraformaldehyde
Analysis software measurement infarct size, calculates the percentage ratio that infarcted cerebral constitution area accounts for the gross area.
6. in cerebral tissue SOD, MDA, NO content mensure
Measure SOD, MDA, NO content in cerebral tissue according to kit specification.The mensure xanthine oxidase of SOD is pressed
Operating procedure is carried out, and at 550 nm, measures absorbance with spectrophotometric.The mensure of MDA presses behaviour with thiobarbituricacidα- method
Carry out as step, at 532 nm, measure absorbance with spectrophotometric.The mensure nitrate reductase method of NO, in 550 nm
Place, measures absorbance with spectrophotometric.
7. statistical procedures
Statistical analysis are carried out using statistics software bag SPSS16.0.Data is used± s represents, compares and checked with t between two groups,
Multigroup is compared and adopts variance analyses.
8. experimental result：
The impact to rats after cerebral ischemic reperfusion cerebral infarct size and Neuroscore for the 8.1 breviscapine extracts
Experimental result shows, model group mice brain infarction area significantly increases, and the lost symptom of serious function of nervous system, with
Sham operated rats relatively have significant difference；The each dosage group of breviscapine all can be effectively improved function of nervous system loss symptom (P ＜
0.05 and P ＜ 0.01), and rat cerebral infarction area can be significantly reduced, compare with significant difference with model group；Wherein small cup flower
The action effect of plain middle dose group, similar with nimodipine matched group.
The impact to rats after cerebral ischemic reperfusion cerebral infarct size and Neuroscore for the table 2 breviscapine extract
| Group | Number of animals | Brain infarction area (%) | Neuroscore |
| Sham operated rats | 10 | 0.00 ± 0.00 | 0.00 ± 0.00 |
| Model group | 10 | 38.8 ± 4.7△△ | 3.42 ± 0.47△△ |
| Matched group | 10 | 27.2 ± 3.5** | 1.93 ± 0.43* |
| High dose group | 10 | 23.6 ± 2.9** | 1.74± 0.29** |
| Middle dose group | 10 | 26.7 ± 5.2** | 2.11 ± 0.31* |
| Low dose group | 10 | 30.9 ± 3.6* | 2.56± 0.29** |
Note:Compare with sham operated rats, △ P<0.05, △ △ P<0.01;
Compare with model group, * P<0.05, * * P<0.01
The impact to rats after cerebral ischemic reperfusion cerebral tissue SOD, MDA and NO content for the 8.2 breviscapine extracts
Test result indicate that, compare with model group, the SOD vigor in rats in sham-operated group cerebral tissue substantially reduces, MDA, NO contain
Amount substantially increases.Compare with model group, the SOD vigor in breviscapine each dosage group rat cerebral tissue substantially increases, wherein, lamp
Small cup florigen is high, middle dose group rat cerebral tissue MDA, NO content substantially reduces；Compare with positive control drug nimodipine group, lamp
Small cup florigen is high, middle dose group rat cerebral tissue MDA, NO content substantially reduces.
The impact (± s) to rats after cerebral ischemic reperfusion cerebral tissue SOD, MDA and NO content for table 3 breviscapine
| Group | Number of animals | SOD (U/ml) | MDA (nmol/ml) | NO(μmol/L) |
| Sham operated rats | 10 | 36.57 ± 7.82 | 1.51 ± 0.49 | 0.82 ± 0.23 |
| Model group | 10 | 13.28 ± 4.64△△ | 2.42 ± 0.57△△ | 3.69 ± 0.53△△ |
| Matched group | 10 | 25.31 ± 8.62** | 2.18 ± 0.61* | 1.91 ± 0.37** |
| High dose group | 10 | 29.18 ± 6.86** | 1.79 ± 0.26** | 1.47 ± 0.26** |
| Middle dose group | 10 | 26.91 ± 7.25** | 1.93 ± 0.38* | 1.86 ± 0.31** |
| Low dose group | 10 | 19.48 ± 5.73* | 2.25 ± 0.29** | 2.52 ± 0.46* |
Note:Compare with sham operated rats, △ P<0.05, △ △ P<0.01;
Compare with model group, * P<0.05, * * P<0.01.
Claims (12)
1. the Preparation method and use of a kind of high-purity breviscapine extract and preparation is it is characterised in that extractive technique bag
Include：Dynamic ultrasound adverse current is extracted or heating and refluxing extraction；Separation process includes：Macroporous adsorbent resin column chromatography, dynamic axial pressure
Contracting (DAC) process-scale chromatography or high speed adverse current chromatogram (HSCCC) separate and prepare.
2. a kind of high-purity breviscapine extract according to claim 1 preparation method it is characterised in that by with
Lower step is realized：Herba Erigerontiss herb is pulverized, plus 6～20 times of volume of water or alcohol that concentration of volume percent is 10%～80% molten
Liquid, or acetone soln, or aqueous isopropanol, dynamic ultrasound adverse current is extracted or heating and refluxing extraction 2～3 times, 0.5～2h every time,
Filter, united extraction liquid, recovered under reduced pressure Extraction solvent, being suitably concentrated into concentration is 0.4～2.0 g crude drug/ml, is centrifuged or filters
Afterwards, plus dilute hydrochloric acid adjust pH to 2.0～7.0 upper prop solution, upper prop solution inhaled by macropore with the flow velocity of 2BV/h～4BV/h
Attached resin chromatography post, upper prop solution is 1 according to the ratio of solid content and dried resin：5～10, first with the distilled water wash of 4～6BV
Resin column bed, elution flow rate is 2BV/h～4BV/h, discards water elution；The alcohol being 10%～80% with concentration of volume percent again
Eluent, elution flow rate is 2BV/h～4BV/h, merges eluent recovered under reduced pressure alcohol, being evaporated to relative density is
1.05～1.30, add the column chromatography silica gel filler of 200～300 mesh, plus methanol mixes post-drying thoroughly, crosses dynamic axial compression reverse
Industrial preparation chromatographic column, methylene chloride-methanol, chloroform-methanol or methanol-water (6：1～2：1) it is eluent gradient eluting, point
The principal piece eluent of breviscapine Shou Ji not contained, merge, concentrating under reduced pressure, add 10 times amount methylene chloride-methanols（8：1～5：1）
Recrystallization, filters, decompression and solvent recovery, lyophilization, spray drying or drying under reduced pressure, obtains final product the high-purity oil lamp of pistac
Florigen（HPLC detects purity >=95%）；
Or take the macroporous resin crude extract of Herba Erigerontiss, add n-hexane-ethyl acetate-methanol/ethanol-water system or just oneself
In the lower phase of alkane-ethyl acetate-n-butanol-water system, concussion makes to be completely dissolved, and carries out high speed adverse current chromatogram（HSCCC）Separate
Preparation；High speed adverse current chromatogram separation condition：Detection wavelength is 285 nm, and main frame temperature is 25 DEG C, n-hexane-ethyl acetate-first
Alcohol-water is solvent system or n-hexane-ethyl acetate-n-butanol-water system（2：8：4：4～6 or 3：7：3～4：6～8）, just
Hexane-ethylacetate（Fixing phase）For upper phase, methanol/ethanol-water or n-butanol-water（Mobile phase）For lower phase；
Fixing phase is filled chromatography column with 10～30 ml/min flow velocitys, adjustment high-speed counter-current chromatograph engine speed is 600
～900 rpm/min, enter mobile phase with the flow pump of 2.0～6.0 ml/min simultaneously, treat biphase reach dynamic equilibrium, open into
Sample valve, sample liquid sample introduction, gathered data simultaneously records ultraviolet chromatogram, reclaims purpose flow point, recovered under reduced pressure under 335 nm wavelength
Solvent, freezing or spray drying, obtain the high-purity breviscapine extract of pistac（HPLC detects purity >=95%）.
3. the preparation method of a kind of high-purity breviscapine extract according to claim 2, Extraction solvent used be water,
Ethanol, methanol, acetone, the isopropanol mixture of one or more therein.
4. a kind of preparation method of high-purity breviscapine extract according to claim 2 is it is characterised in that described
Macroporous adsorbent resin is polystyrene type middle polarity, low pole and nonpolar macroporous adsorption resin, model select ADS-17,
ADS-750、LSA-21、LX-26、XDA-6、D101、D113、D151、D152、D3520、H103、HPD100A、HPD-300、
HPD400、HPD500、HPD-650、HPD-700、HPD-750、HPD-800、HPD-820、DM301、X-5、S-8、SP825、
CAD45,860021, NKA- II or NKA-9 one or more.
5. a kind of preparation method of high-purity breviscapine extract according to claim 2 is it is characterised in that macropore is inhaled
Eluting solvent used by attached resin column chromatography is ethanol-water system or methanol-water system, preferred alcohol-water system；Dynamic axial pressure
Mobile phase used by contracting process-scale chromatography is methylene chloride-methanol, chloroform-methanol or methanol-water system (6：1～2：1) gradient elution；
Eluting solvent used by high speed adverse current chromatogram be n-hexane-ethyl acetate-methanol/ethanol-water system or n-hexane-ethyl acetate-
N-butanol-water system.
6. the Preparation method and use of a kind of high-purity breviscapine extract according to the preparation of claim 1 method and preparation,
It is characterized in that, make medically acceptable various pharmaceutical preparatioies such as by adding various pharmaceutic adjuvants：Conventional tablet, thin film
The solid orally ingestibles such as coated tablet, capsule, dispersible tablet, drop pill, granule, sustained-release preparation, and spray, aerosol
The dosage forms such as agent, freeze-dried powder, injection.
7. the Preparation method and use of a kind of high-purity breviscapine extract according to claim 6 preparation and preparation, it is special
Levy and be, in preparation the mass percent of principal agent be 10%～80%, pharmaceutically acceptable adjuvant mass percent be 20%～
90%.
8. a kind of high-purity breviscapine extract formulation according to claim 6, including：Conventional tablet, film coating
Piece, dispersible tablet, slow release or controlled release tablet it is characterised in that described label by 10%～70% mass percent principal agent, 10%～
The dilution adjuvant of 60% mass percent, the slow release of 10%～50% mass percent or controlled-release excipient, 1%～15% mass percent
Disintegrate adjuvant, the bonding adjuvant of 1%～20% mass percent, 0.3%～5% mass percent lubrication adjuvant composition.
9. high-purity breviscapine extract formulation according to claim 6 dilutes adjuvant it is characterised in that label is used
Mixture for one or more of starch, Lactose, dextrin, sucrose, Microcrystalline Cellulose, calcium hydrogen phosphate, calcium sulfate；Disintegrate is auxiliary
Material refers to carboxymethyl starch sodium (CMS-Na), low-substituted hydroxypropyl cellulose (L-HPC), cross-linking sodium carboxymethyl cellulose (cCMC-
Na), the mixture of one or more of crospolyvinylpyrrolidone (PVPP), Crospovidone, hydroxypropyl starch；Bonding
Adjuvant refers to Microcrystalline Cellulose, hydroxypropyl methylcellulose ketone（HPMC）, sodium carboxymethyl cellulose（CMC-Na）, percent by volume be
50%～95% Polyvinylpyrrolidone (PVP K25) ethanol solution, starch slurry, the mixture of one or more of syrup；Lubrication
Adjuvant refers to the mixture of one or more of Pulvis Talci, magnesium stearate, micropowder silica gel.
10. high-purity breviscapine extract formulation according to claim 6 is it is characterised in that the outward appearance of described tablet
For circular piece or special-shaped tablets, including：Triangular piece, ellipse slice, losenges.
11. high-purity breviscapine extract formulations according to claim 6 are it is characterised in that the base of described drop pill
Matter is poloxamer-188 (Poloxamer-188), PEG-4000 (PEG-4000), PEG-4000 (PEG-
6000), the mixture of one or more of xylitol, medical starch, sodium stearate, polyoxyethylene monostearate, instills
Coolant is the mixture of one or more of dimethicone, liquid paraffin, vegetable oil；
Drop pill preparation condition：Medicine and substrate composition are 1：4～1:2,70 DEG C～90 DEG C of fluid temperature, take insulation dripping, drip
Head bore（Inside/outside footpath）：2.1/2.5 mm～3.0/5.0 mm, drips speed：20 d/min～30 d/min, drip away from：4～10 cm.
12. according to claim 1 method preparation a kind of high-purity breviscapine extract and its preparation, to cardiovascular and cerebrovascular vessel disease
Disease, such as：Coronary heart disease, myocardial infarction, angina pectoriss, obliterative vascular disease, vasculitiss etc.；Cerebral blood supply insufficiency, apoplexy sequela,
The acute stage of ischemic cerebrovascular, convalescent period and sequela, such as：Sequela paralysis caused by cerebral thrombosiss, cerebral hemorrhage, dementia
Deng there being significant curative effect.
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